Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

Oral fluids provide ready detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses. This study sought to evaluate relationships between oral virus, oral and systemic anti-SARS-CoV-2-specific antibodies, and symptoms. Oral fluids (saliva/throat wash (saliva/TW)) and serum were collected from asymptomatic and symptomatic, nasopharyngeal (NP) SARS-CoV-2 RT-qPCR+ human participants (n = 45). SARS-CoV-2 RT-qPCR and N-antigen detection by immunoblot and lateral flow assay (LFA) were performed. RT-qPCR for subgenomic RNA (sgRNA) was sequence confirmed. SARS-CoV-2-anti-S protein RBD LFA and ELISA assessed IgM and IgG responses. Structural analysis identified host salivary molecules analogous to SARS-CoV-2-N-antigen. At time of enrollment (baseline, BL), LFA-detected N-antigen in 86% of TW and was immunoblot-confirmed. However, only 3/17 were saliva/TW qPCR+ . Sixty percent of saliva and 83% of TW demonstrated persistent N-antigen at 4 weeks. N-antigen LFA signal in three anti-spike sero-negative participants suggested potential cross-detection of 4 structurally analogous salivary RNA binding proteins (alignment 19–29aa, RMSD 1–1.5 Angstroms). At enrollment, symptomatic participants demonstrated replication-associated sgRNA junctions, were IgG+ (94%/100% in saliva/TW), and IgM+ (63%/54%). At 4 weeks, SARS-CoV-2 IgG (100%/83%) and IgM (80%/67%) persisted. Oral and serum IgG correlated 100% with NP+ PCR status. Cough and fatigue severity (p = 0.010 and 0.018 respectively), and presence of weakness, nausea, and composite upper respiratory symptoms (p = 0.037, 0.005, and 0.017, respectively) were negatively associated with saliva IgM but not TW or serum IgM. Throat wash IgM levels were higher in women compared to men, although the association did not reach statistical significance (median: 290 (female) versus 0.697, p = 0.056). Important to transmission and disease course, oral viral replication and persistence showed clear relationships with select symptoms and early oral IgM responses during early infection. N-antigen cross-reactivity may reflect mimicry of structurally analogous host proteins.


Enrollment
A total of 47 participants were assessed May through August of 2020.Inclusion criteria for the symptomatic longitudinal observational cohort (n = 15) required participants to be NP SARS-CoV-2 RT-qPCR positive.NP+ status was recorded by testing at UNC McClendon Hospital labs.COVID-19+ patients were enrolled within 7 days of their positive NP test after written informed consent and were stratified with mild, moderate or severe symptoms at the time of presentation based on the NIH criteria (NIH COVID-19 treatment guidelines).For the asymptomatic cohort (n = 30), participants were either asymptomatic, anti-SARS-CoV-2 spike seronegative (n = 15) or asymptomatic, SARS-CoV-2 spike seropositive (n = 15).These participants were UNC-affiliated students, staff and faculty who provided written informed consent.Archived, pre-COVID pandemic saliva (n = 5) and throat wash samples (n = 2) from 2014 (UNC IRB study #20-0792) were used as SARS-CoV-2 negative controls.All samples were obtained and stored under IRB approval (Symptomatic participants, UNC IRB study #20-0792; Asymptomatic participants, UNC IRB study #20-1771; Archived, PreCOVID participants, UNC IRB study #07-1431).All experimental protocols used to assess specimens were performed in accordance with approval by the University of North Carolina IRB (Symptomatic participants, UNC IRB study #20-0792; Asymptomatic participants, UNC IRB study #20-1771; Archived, PreCOVID participants, UNC IRB study #07-1431).All methods used to assess specimens were performed in accordance with the relevant guidelines put forth by the University of North Carolina at Chapel Hill.

Biospecimens
For RT-qPCR, saliva and throat wash samples were collected concurrently.The NPS samples were collected in 3 ml of viral transport medium (VTM).Unstimulated whole mouth fluid (WMF) samples (saliva) and throat wash gargle with 10 ml of normal saline (throat wash) were each collected in sterile 50 mL wide-mouthed screwcapped containers.Whole blood samples were collected by venipuncture or finger prick in standard EDTA tubes.The whole blood samples were centrifuged within 24 h of collection at 1600 g for 30 min to separate plasma.Prior to serological testing, all plasma specimens were heat-inactivated at 56 °C for 30 min, mixing the sample by inverting the tube every 5 min.The inactivated samples were centrifuged at 1500 g for 10 min, and the supernatant was aliquoted and stored at − 80 °C until further testing.Samples were collected in the following order: NP, then throat wash, saliva, and blood.Samples were immediately transported for processing where the saliva, throat wash, and blood samples were stored at 4ºC and processed within 24 h.For oral samples, processing include centrifugation at 1000xG for 10 min, supernatant was removed and pellets were stored separately.After processing 1 ml aliquots of samples were stored at -80 °C.Similarly, 1 ml aliquots of archived, preCOVID-19 saliva and throat wash samples have been stored at − 80 °C for > 5 years.

Nucleic acid extraction and RT-qPCR
Saliva and throat wash from COVID-19 patients were used as a source for the detection of SARS-CoV-2 RNA.Trizol (Life Technologies, Carlsbad, CA) was used to inactivate virus and RNA was extracted according to the manufacturer's instructions.Briefly, 750 µl of Trizol were added to 250 µl of oral fluid (saliva or throat wash).Following chloroform addition and phase separation, the aqueous phase was collected.Glycogen (2 µg) was added to the aqueous phase and RNA was precipitated with isopropanol.RNA pellets were dried and resuspended in water.
RNA was reverse transcribed using Superscript III (Life Technologies) according to the manufacturer's instructions.First strand synthesis was primed with random hexamers (Life Technologies).First strand synthesis reactions were used in SybrGreen-based qPCR.Three regions of the SARS-CoV-2 genome were targeted using virus-specific primers: WTSorf1F/WTSorf1R, E_Sarbeco_F/E_Sarbeco_R 12 and 2019-nCoV_N1-F/2019-nCoV_N1-R 13 .A standard curve was generated using SARS-CoV-2 reference RNA (ATCC VR-3276SD) for absolute copy number quantitation.
Subgenomic RNA (sgRNA) detection was performed using the forward primer, CoV25UTR (nucleotides 1-25; GenBank:MN908947) with the reverse primers, SRTqPCRR, orf3aRTqPCRR or 2019-nCoV_N1-R .Percent sgRNA was calculated using the equation: 2 − (sgRNA Ct − N Ct) × 100 where sgRNA Ct values were signal obtained using the 5'UTR/specific sgRNA primer pair and the N Ct value was signal obtained using the 2019-nCoV_ N1-F/2019-nCoV_N1-R primer pair.Following qPCR, reactions from positive wells were removed, treated with ExoSapIt according to the manufacturer's instructions (USB, Cleveland, Ohio) and Sanger sequencing was performed (Eton Bioscience, Research Triangle Park, NC) using gene-specific primers: SRTqPCRR for SsgRNA , orf3aRTqPCRR for orf3asgRNA or 2019-nCoV_N1-R for NsgRNA.Total RNA was isolated from the saliva of SARS-CoV-2 -infected individuals.RT-qPCR was used to amplify cDNA encoding SARS-CoV-2 Spike (S), Envelope (E) or N-antigen.The generated cDNAs were digested with SfiI and SalI and cloned into SfiI/SalI sites of pCMV-myc eukaryotic expression plasmid.Expression in eukaryotic cells would result in the production of viral proteins with an amino-terminal c-myc tag.Immortalized human oral keratinocytes (NOK) were grown in keratinocyte serum-free media (Life Technologies, Carlsbad, CA).Cells were transfected with expression vectors using Fugene-6 (Promega, Madison, WI).Forty-eight hours post transfection, media was removed and subjected to SDS-PAGE and immunoblot analysis to detect myc-tagged proteins.
To obtain lysates from whole saliva, TW or transfected NOK, protein was isolated from the organic phase obtained using Trizol (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions.Protein was precipitated from half of the organic phase after the addition of 750ul isopropanol and centrifugation at 12,000 X G. Pellets were washed twice with 95% ethanol/0.3M guanidine-HCl followed by a final wash with 100% ethanol.Each wash was performed for 20 min.Final protein pellets were dried and resuspended in 1X sample buffer (250 mM Tris, 8.0, 2% SDS, 20% glycerol, 50 mM DTT, 0.1% bromophenol blue).Pellets in buffer were incubated at 65° C until dissolved.

Immunoblot analysis of NOK cell lysates and media
Twenty-five micrograms of whole lysate or 10 µl of transfected cell culture media were loaded onto precast NuPage 4-12% Bis-Tris gels (Life Technologies, Carlsbad, CA).Proteins were electroblotted to PVDF membrane using western blot transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) for 2 h at 200 mamps, constant current.Following transfer, blots were blocked by incubation with 5% nonfat dry milk in 1X PBS containing 0.1% Tween-20 (1X PBS-T) at room temperature.Following blocking, blots were incubated with primary antibody overnight at 4° C. Blots were washed twice with 1X PBS-T followed by incubation with HRP-conjugated secondary antibody (Promega) diluted 1:10,000 in PBS-T, 5% milk for 1 h at room temperature.Blots were washed twice with PBS-T and protein bands were detected by ECL after incubation with ECL Prime (GE Healthcare, Chicago, Il) according to the manufacturer's instructions.Blots were imaged using a GE ImageQuant LAS4000 system.The following primary antibodies were used to detect proteins on western blots: rabbit anti SARS-CoV-2 nucleocapsid (PA5-114,448, Life Technologies), mouse anti-c-myc (sc-40, Santa Cruz Biotechnology, Dallas, TX), anti-amylase (A8273, Sigma-Aldrich, St. Louis, MO).

Overexpression of His-tagged SARS-CoV-2 nucleocapsid in E.coli
The NcoI/SalI fragent from pCMV-mycN containing the entire coding region of N was removed and inserted into the NcoI/SalI sites of pET30a.The resulting plasmid, containing the SARS-CoV-2 nucleocapsid (N) coding region in frame with the 6X histidine tagged coding region of pET30, was used to transform E. coli BL21 (DE3) bacteria.Bacteria with pET30a empty vector were used as a source of negative control his-tagged protein.Bacteria containing pET30a will produce an ~ 9 kD his-tagged protein after induction with IPTG.Single colonies were grown in LB medium containing kanamycin (50 µg/ml) to an OD A600 of 1.0.To induce gene expression, IPTG (1 mM final concentration) was added and cultures were incubated at 30° C for 2 h.Following centrifugation of cultures, bacterial pellets were resuspended in 1 ml NPI-10 (300 mM NaCl, 50 mM sodium phosphate, pH8.0, 10 mM imidazole, 1 mg/ml lysozyme).Lysates were incubated with NTA-Ni agarose beads (Life Technologies) to purify his-tagged proteins.Beads were washed with NPI-20 (300 mM NaCl, 50 mM sodium phosphate, pH8.0, 20 mM imidazole).Protein was eluted by resuspending beads in 250 µl NPI-500 (300 mM NaCl, 50 mM sodium phosphate, pH8.0, 500 mM imidazole).Eluates were subjected to SDS-PAGE followed by Coomassie staining to assess retrieval and protein purity.Protein concentration was determined by Bradford assay.

Lateral flow assay
Detection of SARS-CoV-2 N-antigen or anti-SARS-CoV-2 spike protein IgG and IgM was accomplished using lateral flow chambers (BioMedomics, Research Triangle Park, NC).For N-antigen 50 µl of sample (saliva or throat wash) was diluted with 50 µl of lysis buffer (supplied by the manufacturer).Samples in lysis buffer were incubated at room temperature.Following incubation, 80 µl of sample was applied to the lateral flow chamber.Bands were visualized and quantitated using ImageJ software.The control band for each detection cartridge was used to normalize antigen-specific band.For detection of anti-spike IgG/IgM, 50 µl of sample (saliva or throat wash) was added directly to the lateral flow chamber.Two drops of COVID-19 IgG/IgM rapid test buffer (supplied by manufacturer) were added and bands were allowed to develop.Control bands were used normalize IgG and IgM intensity.Quantitation was determined relative to signal produced by pre-COVID-19 archived saliva and throat wash samples.

Serology
Serum anti-SARS-CoV-2 spike protein IgG and IgM were detected by ELISA as previously described 14 .

NCBI Structural analysis
The crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 N antigen (PDB ID: 6M3M) was compared to other publicly available crystal structures in the Molecular Modeling Database (MMDB) using Vector Alignment Search Tool (Vast+) within the Domains and Structures module on the National Center for Biotechnology Information website (https:// www.ncbi.nlm.nih.gov).Vast+ makes geometric structural comparisons between macromolecules in the absence of sequence similarity.The 3D structures of superimposed biological assemblies were visualized using the web-based 3D viewer iCn3D version 4.3.1 to support the visualization style 15 .

Statistical methods
Throughout, we applied two-sided non-parametric exact tests, each with a 0.05 type I error rate and no adjustment for multiplicity.Associations between ordinal symptom severity and quantitative variables (either N-antigen or antibody level) were evaluated using an exact Kendall rank correlation test.Quantitative variables were compared between two independent groups using an exact Mann-Whitney U test.Within-participant changes in a quantitative variable (N-antigen or antibody level) across two timepoints (baseline vs. day 28) were evaluated using an exact Wilcoxon signed-rank test.Baseline (date of enrollment) measurements were conducted within 7 days of study enrollment (i.e., within one week of the positive NP SARS-CoV-2 test).

Results
Assessment of the SARS-CoV-2 N-antigen lateral flow assay N-antigen LFA was validated using lysates from immortalized normal oral keratinocytes (NOK) transfected with a myc-tagged SARS-CoV-2 N-antigen expression vector.N-antigen was detected by anti-myc antibody immunoblot (Fig. 1A) or LFA (Fig. 1B).Bacteria-produced, his-tagged N-antigen was purified (Fig. 1C), used for N-LFA quantification, and migrated at 55 KD, matching the predicted molecular weight of SARS-CoV-2 N-antigen, while pET30 vector negative control ran at ~ 9 KD (Fig. 1C).LFA limit of detection was determined using two-fold serial dilutions of preCOVID-19 saliva containing recombinant his-tagged N-antigen at levels ranging from 43 to 680 picograms (pg).A faint band at 85 pg and robust detection > 170 pg demonstrates LFA's semi-quantitative nature (Fig. 1D).No band was detected in saliva spiked with pET30 recombinant protein, indicating signal specificity.LFA allowed longitudinal detection of N-antigen in saliva from a representative NP-RT-qPCR+ participant at symptom onset 14, and 28 days, suggesting active persistent infection, while signal was undetected in two PreCOVID-19 saliva samples (Fig. 1E).

Assessment of the SARS-CoV-2 Anti-Spike RBD IgM/IgG lateral flow assay
Recombinant anti-Spike RBD IgG/IgM in LFA assays detected anti-SARS-CoV-2 Spike-specific IgM and IgG oral immune responses.Two-fold dilution allowed immunoglobulin LFA validation.Mixtures of Spike RBDspecific recombinant human IgM/IgG in preCOVID-19 saliva detected analytical sensitivity down to 0.63 µl for IgM, however, IgG was undetected after a single twofold dilution (Fig. 1F).Archived saliva, spiked with anti-SARS-CoV-2 antibody demonstrated IgM signal but not IgG, perhaps reflecting IgM detection of a pre-2019 human coronavirus.Unspiked demonstrated no signal (Fig. 1G).A symptomatic participant demonstrated IgG at baseline and at 14 days but not IgM (Fig. 1G).

Viral metrics in the oral fluids over time and between sexes
LFA test and control band intensity was measured by ImageJ to provide relative measures of N-antigen and immunoglobulin presence in oral fluids and by ELISA for serum (Fig. 6A).While less N-antigen was detected in symptomatic TW at 28 days compared to baseline (relative band intensity (RBI), median difference (28D-Baseline): − 44.604 RBI), this decrease was not statistically significant (Wilcoxon Signed-Rank Test, p = 0.875).In the symptomatic group, differences in TW N-antigen were not detected between preCOVID-19 archived saliva vs baseline, (Wilcoxon Rank Sum Test, p = 0.103).(Fig. 6B, 6B supplement).At baseline, 56% of the symptomatic were N-antigen positive in saliva and 91% were N-antigen positive in TW by LFA.Using concurrently collected NP and oral samples NP-RT-qPCR served as the gold standard.LFA assay sensitivity was 0.533 (95% CI, 0.266, 0.787) in saliva and 0.909 (CI, 0.587,0.998) in TW (Fig. 6C).Salivary IgG levels were different between baseline and preCOVID-19 archived samples (Mann-Whitney U test, p = 0.002).Changes over time were not detected in salivary IgM, serum IgG, or serum IgM. (Fig. 6A, 6A Supplement).While sex differences were not detected in oral N-antigen or TW IgG, women appeared to have higher levels of TW IgM than men (median: 290 (female) vs. 0.697 (male), Mann-Whitney U test, p = 0.056) (Fig. 6D).

Discussion
In this study, we consistently detect SARS-CoV-2 antigen and antibody in oral fluids during symptomatic COVID-19.N-antigen detection was immunoblot-and sgRNA-confirmed in NP-RT-qPCR+ participants providing significant implications for oral transmission.SARS-CoV-2-targeted oral IgG responses were highly correlated with nasopharynx positivity and oral N-antigen detection.Salivary IgM levels indicated both symptom presence and severity.
This study, and others, detected salivary SARS-CoV-2 RNA with distinct viral shedding dynamics compared to NP 1,18 .Prior assessments detected a threefold lower positive detection rate in saliva than NPS 18 , leading some to question the role of oral virus.Here, cumulative data suggest oropharyngeal viral replication.SARS-CoV-2 was detected in oral fluids from NP-RT-qPCR+ participants using several RT-qPCR-based viral detection methods, (1) three distinct primer pairs targeting 3 regions of the viral genome, (2) absolute RNA copy number determination using a standard curve, and (3) sgRNA, shown to be a marker of active replication during early symptomatic infection 19,20 .Orf3A sgRNA was detected in 82% of those tested by sgRTqPCR, (fast, sensitive, economically feasible, and reliable) and is a viable marker of viral replication based on its contribution to viral titer and disease in hACE+ mice 21 .SARS-CoV-2 protein was consistently detected by two distinct methods, LFA and immunoblot.We are unaware of other studies demonstrating immunoblot confirmed viral antigen detection in oral fluids.Importantly, persistent N-antigen detection provides significant implications for continued potential oral transmission (Figs.3F, 8B).
One critique of rapid LFA was false positives.Here, we detect positive LFA results in two seropositive (1A,4A), and three uninfected participants (16A,17A,18A).During asymptomatic infection it is impossible to determine where participants are in their infection cycle.Seropositive/NP-participants could demonstrate oral infection (1A,4A) or oral infection may subside in seropositive/NP+ (8A).Alternatively, cross-reactive host proteins might be detected.Analysis determined potential cross-reactivity with structurally analogous host RNA binding proteins (Fig. 5).17S U2 snRNP and RBM7, are salivary proteome members and two others, LINE1 Orf1p and hnRNP H, have salivary proteome isoforms (LINE1 Orfp1 and hnRNP A2/B1, hnRNP M, hnRNPK) 22 .This raises the possibility that N-antigen-specific LFA can detect conformationally-similar proteins, perhaps reflecting the potential for N-antigen mimicry through RNA binding domains.An example implication is SARS-CoV-2 host genome integration.The LINE1 ORF1p analogue mediates retrotranposable element genome integration 23 .RNA virus sequences have been detected across vertebrate genomes, with several integration signals consistent with LINE retrotransposon germline integration of viral cDNA copies 24 .Recently, subgenomic sequences, derived from SARS-CoV-2's 3' end, were shown to be integrated into host cell DNA 25 .Hence, host interactions with N-antigen or similar actions by N-antigen might facilitate SARS-CoV-2 genome integration.
Anti-spike RBD-specific antibodies during natural infection and vaccination were detected in saliva 26,27 with temporal kinetics that reflect systemic immune responses.While anti-spike-RBD IgG levels were negatively correlated with salivary viral load in the Silva study 28 , here in 15/15 symptomatic and 1/1 asymptomatic participants, anti-spike-RBD IgG was 100% positively correlated with SARS-CoV-2 NP-RT-qPCR+ status, and symptomatic individuals consistently demonstrated salivary N-antigen by immunoblot.The NP+ status and saliva IgG relationship was also shown by Pisanic et,al.with IgG positive responses in 24/24 RT-qPCR-confirmed COVID-19 cases 11 .Together, this suggests that salivary IgG detection indicates concurrent nasopharynx positivity.Detection of simultaneous oral virus and oral host immune responses at baseline suggests a highly infectious presymptomatic state followed by concurrent symptoms, persistent oral infection, and virus-targeted host responses.
Oral fluids hold promise as SARS-CoV-2 prognostic indicators.Huang et al. suggested a correlation between salivary viral RNA burden and COVID-19 symptoms, including taste loss 1 .Others showed positive associations between salivary viral load (VL) and COVID-19-related pro-inflammatory markers; IL-6, IL-18, IL-10, and CXCL10 28 .Here, oral viral RNA detection did not correlate with COVID-19 symptoms.Relationships between IgG, VL, and disease course were not detected 18,29 .However, we detected associations between salivary IgM and multiple symptoms including weakness, nausea, and upper respiratory symptoms.Of interest, these trends were not detected in serum IgM in the same participants.Relative IgM levels in saliva were associated with degree of fatigue and cough severity suggesting that oral IgM signifies an active early immune response associated with milder disease course.
Sex differences are detected in COVID-19-related morbidity and mortality 30 .Here, sex differences in serological response were detected, with women having numerically higher oral IgM levels than men.Systematic review of sex differences determined that women were less likely to present with severe disease and be admitted to the intensive care unit (ICU) than men (OR 0.75 [0.60-0.93]p < 0.001 and OR 0.45 [0.40-0.52]p < 0.001, respectively 31 .While higher smoking rates and reluctance to seek health care may contribute to male disease, the well-described evolution of greater humoral immunity in females could contribute to these differences 32,33 .Here, women tended to have higher IgM levels, and individuals with higher IgM demonstrated fewer symptoms, and milder symptom severity.Differences in IgM production may provide biologic underpinning, contributing to the COVID-19-related morbidity and mortality sex gap. These findings provide novel insights to oral SARS-CoV-2 infection.Oral IgM detection predicted milder disease in terms of symptoms and severity.The potential for cross-detection of N-antigen and structurally Figure 3. Oral virologic characterization of study participants at baseline.All participants were assayed by qPCR for detection of viral RNA in the nasopharynx.Saliva/TW was assayed by qPCR for viral RNA and for LFA-based detection of N-antigen, anti-spike IgG and anti-spike IgM.(A) Oligonucleotide sequences for primers used for RT-qPCR and production of cDNA for construction of expression plasmids.E_Sarbeco_F and E_Sarbeco_R have been described.The sequences of 2019-nCoV_N1-F and 2019-nCoV_N1-R, were obtained from the CDC.The SARS-CoV-2 reference sequence GenBank:MN908947 was used to determine oligonucleotide sequences and map coordinates.SfiI (ggccatggaggcc) and SalI (gtcgac) restriction enzyme sites used for cloning into the expression vector, pCMV-myc, are underlined in expression primers.The start codon and stop codons in expression primers are in bold.(B) Standard curves were generated by RT-qPCR using know copy numbers of SARS-CoV-2 RNA and primers targeting the nucleocapsid (N) coding region to quantitate viral RNA isolated from patient saliva and TW.(C) Immunoblot was used to detect SARS-CoV-2 structural proteins in cell culture media obtained from NOKs transfected with individual expression vectors encoding myc-tagged, SARS-CoV-2 structural proteins (S, E, M and N) or cotranfected with all myc-tagged expression vectors.Medium from cells transfected with empty vector (pCMV-myc) was used as a negative control.Proteins were detected using myc-specific antibody (mouse anti-myc, sc-40, Santa Cruz Biotechnology).(D, E).Protein from subset of saliva and TW samples (respectively) was used for the detection of viral proteins.Immunoblot detection of SARS-CoV-2 N-antigen was assessed in a subset of symptomatic individuals (n = 10) at baseline (top panel) using N-specific antibody (PA5-114,448, LifeTechnologies, Inc).Detection of salivary alpha-amylase (anti-amylase, A8273, Sigma-Aldrich) served as a loading control (bottom panel).Saliva or TW from archived preCOVID-19 patients A and B were included as negative controls.Cell culture medium from NOK cells transfected with myc-tagged SARS-CoV-2 N-antigen expression construct was used as a positive control.Cell culture medium from NOKs transfected with empty vector was included as a negative control.N-antigen was detected at 55 KD and alpha amylase, the loading control, was detected at 58.4 KD. (F, G) Table and graph of symptomatic participants (n = 17) who were all nasopharynx RT-qPCR positive.RT-qPCR and LFA was also performed on saliva and TW of these participants, only RNA was available for participant 0517.Spike ELISA was performed on serum (SER).Viral RNA copy number (molecules of virus RNA) in 25 µl of saliva or TW was determined using N-specific primers.Green highlight indicates a positive result.Red highlight indicates a negative result.ND indicates not determined.(H) Asymptomatic participants (n = 15) were all anti spike IgG positive in their serum.(I) COVID-19 negative participants (n = 15) were anti-spike negative and nasopharynx negative.

Figure 1 .Figure 2 .
Figure 1.Assessment of Lateral flow assays for detection of SARS-CoV-2 N-antigen and anti-SARS-CoV-2 spike RBD domain targeted antibodies (A) Expression of myc-tagged N-antigen in NOK cells by immunoblot.Vector lane indictates cells transfected with the empty plasmid, pCMV-myc.N-antigen detected with antimyc antibody is indicated by the arrow.(B) LFA detected N-antigen in the lysates of NOKs transfected with myc-tagged N expression plasmid.Both, control and N-antigen bands, are indicated.Only the control band is detected in lysates from cells transfected with empty vector.(C) Expression and purification of His-tagged SARS-CoV-2 N-antigen in E.coli.A band consistent with the size of N-antigen (55 kD) can be seen in crude lysates from cells transformed with His-tagged, N-antigen expression plasmid.This band is eluted from Ni-beads during purification.His-tagged protein expressed in bacteria transformed with empty vector (pET30) served as a negative control.(D) Dilution series of His-tagged N-antigen in preCOVID-19 saliva and detection using Biomedomics N-antigen LFA.(E) Detection of N-antigen in saliva of preCOVID-19 and symptomatic SARS-CoV-2 NP+ participants at baseline and post baseline 14D and 28D by LFA.The detection of N-antigen band is indicated.(F) Two-fold dilution series of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 saliva and detection using Biomedomics IgG/IgM lateral flow assay cartridges.Control, anti-S-RBD IgM and S-RBD IgG are indicated.The 0 lane indicates unspiked, preCOVID-19 saliva only.(G) Detection of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 or a symptomatic SARS-CoV-2 NP+ subject at baseline and post baseline 14D saliva using Biomedomics IgG/IgM lateral flow assay cartridges.

Figure 4 .
Figure 4.The SARS-CoV-2 nucleocapsid mimics host RNA binding proteins that are expressed within the salivary proteome and may be responsible for cross reactivity in LFA assays.VAST+ was used to generate RNA binding protein structures demonstrating 3D similarity to the SARS-CoV-2 N-antigen.Simultaneous alignment generated molecular protein pairs between N-antigen (first column) and host RNA binding proteins (second column).Non-aligned N-antigen amino acids are rendered in green, conserved aligned amino acids are rendered in red and aligned, non-conserved amino acids are rendered in blue.Non-aligned host protein amino acids proteins are rendered in olive green, conserved aligned amino acids are rendered in pink and aligned, non-conserved amino acids are rendered in light blue.The spatial arrangement of overlapping amino acids can be seen in the merged sequence alignment (third column).The number of structurally aligned amino acids (conserved and non-conserved) is shown.The root mean square deviation (RMSD) is used as a measurement between atoms in the backbone of the molecular structures and is listed in angstroms above each merged image and detection of the host protein within the salivary proteome is displayed as yes in the fourth column while detection of a related isoform within the salivary proteome is displayed as ISO.

Figure 5 .
Figure 5. SARS-CoV-2 subgenomic RNAs (sgRNA) are detected the saliva of symptomatic COCID patients.(A) Schematic diagram showing the RNA genome of SARS-CoV-2 (~ 30 kb) and the subgenomic RNAs encoding major viral structural proteins (S, E, M and N) as well as orf3a.Detection of SARS-CoV-2 RNA by RT-qPCR was performed using primer pairs corresponding to orf1 (blue arrows), E (peach arrows) and N (red arrows) coding regions.SgRNA was detected using a forward primer corresponding to the 5'UTR (black arrow) and either S-specific (yellow), orf3a-specific (green) or N-specific (red arrow) reverse primers.The RT-qPCR signal generated with N1 primers (red arrows) were used to calibrate levels of sgRNA.(B, C, D) The percent levels of S, orf3a or N sgRNA determined using the 5'UTR primer and the specific sgRNA reverse primer are shown.Percent sgRNA levels relative to total viral RNA levels are determined as follows: 2^-(sgRNA Ct/N Ct) × 100.Direct sequencing of RT-qPCR amplified products was performed to determine the junction sequence of the 5'UTR and the coding region of each sgRNA.Sequencing information obtained from samples is indicated with an asterisk.The overlap sequence between the 5'UTR and each specific sgRNA is indicated.

Figure 6 .
Figure 6.Longitudinal analysis of SARS-CoV-2 N-antigen and anti-SARS-CoV-2 specific IgG/IgM antibodies in oral fluids of infected individuals demonstrates persistence and sex differences (A) Detection of N-antigen in Saliva (Sal), anti-Spike IgG and anti-Spike IgM in Saliva (Sal) and Serum of symptomatic participants, at baseline (BL), 14 days (14D), and 28 days (28D) post baseline compared to preCOVID-19, archived (Pre) TW. (B) LFA detection of N-antigen in TW of symptomatic participants at baseline (BL) and 28 days (28D) post baseline compared to preCOVID-19, archived (Pre) TW.The Mann-Whitney U test assessed difference between preCOVID-19 and baseline and the Wilcoxon Signed-Rank Test assessed differences between paired baseline and 28-day samples.(C) Saliva and TW N-antigen, anti-Spike IgG and anti-Spike IgM LFA assay sensitivity was determined relative to NP-RT-qPCR+ results in symptomatic participants.(D) Detection of N-antigen in TW, anti-Spike IgG and anti-Spike IgM in TW and Serum of female vs male symptomatic participants at baseline.

Figure 7 .
Figure 7. Salivary IgM predicted a more favorable disease course.The relationship between cough and fatigue symptom severity and SARS-CoV-2 protein and anti-spike antibody levels in saliva (Sal) and TW of symptomatic infected individuals was assessed at baseline using LFA.The Kendall Rank Correlation test detected a decreasing relationship between salivary IgM levels and the severity of fatigue and cough.

Figure 8 .
Figure 8. Salivary IgM was associated with the absence of most symptoms.(A) The relationship between symptom presence (weakness, muscle ache, nausea, loss of taste and smell and upper respiratory symptoms [including cough, shortness of breath, sore throat, nasal obstruction (stuffy nose), nasal discharge [runny nose]) and semi-quantitative SARS-CoV-2 N-antigen in saliva (abbreviated Sal) and TW, and anti-spike antibody levels in saliva (abbreviated Sal), TW, and Serum of symptomatic infected individuals was assessed at baseline as determined by LFA.The Mann-Whitney U test detected a relationship between the absence of symptoms and higher oral IgM levels in saliva.(B) Longitudinal oral virologic characterization of symptomatic participants detects viral persistence with concurrent antibody responses suggesting presentation post-acute infection.Saliva (triangle), TW (square), and Serum (circle) were assayed by LFA for detection of N-antigen (brown), anti -spike IgG (green) and anti-spike IgM (blue) at baseline, 14d and 28d (n = 15).